Human cytomegalovirus (HCMV) is a ubiquitous member of the herpesvirus family that can induce a wide range of diseases, typically in newborns and immunocompromised adults. Nearly one percent of all live births in the United States are associated with congenital HCMV infection, with approximately 5 to 10 percent of infections resulting in significant neurological defects. In bone marrow transplant recipients, mortality due to HCMV pneumonia can be as high as forty percent. In addition, disseminated HCMV infection is common in AIDS patients and is frequently associated with conditions such as gastroenteritis and sight-threatening chorioretinitis.
The viral genome consists of a large double-stranded DNA molecule of approximately 230 kilobase pairs packaged within an enveloped capsid to form the infectious virion. Productive infection is species- and cell-specific and requires the tightly coordinated sequential expression of viral genes. Viral genes are divided into three kinetic classes: immediate early (IE), early (E) and late (L). The IE gene products, regulated by a complex enhancer promoter, are synthesized immediately after entry of the viral genome into the nucleus of infected cells and rely primarily on host factors for their expression. Transcriptional regulation of IE genes has been extensively studied and three major IE proteins have been characterized: IE72, IE86 and IE55. Early genes are transcribed prior to viral DNA replication. The late genes, which constitute the majority of the viral genome, are transcribed in abundance only after viral DNA replication. Both early and late gene expression is modulated by one or more viral IE proteins, as well as host proteins.
Studies of the biological and biochemical function of IE72, IE86 and IE55 have indicated that these proteins play a critical role in HCMV cascade gene expression. All of these proteins have been shown to be involved in the transactivation of HCMV early promoters, as well as heterologous viral and cellular promoters. IE86 also plays a major role in repressing its own promoter, the major immediate early promoter (MIEP). The IE72 and IE55 proteins act to enhance the activity of the MIEP and augment the stimulatory effect of the IE86 protein on its responsive promoters.
Recently, the IE86 protein was shown to enhance UL112 early promoter activity by binding to discrete sequences. Three IE86 binding sites were identified in this promoter. However, direct binding of IE86 to the promoter is not absolutely required because deletion of these target sites retained 40% of the response to IE86 transactivation (Arlt et al., J. Virol. 68:4117-4125, 1994). This transactivation by IE86 appears to involve the interaction of IE86 with the cellular transcriptional factor CREB (Lang et al., J. Virol. 69:6030-6037, 1995), which differs from the mechanism of transactivation of the HCMV early promoter UL54 (DNA polymerase, pol). An expression construct encoding the major IE proteins IE72, IE86 and IE55 has been shown to induce transactivation of the pol promoter (see Stenberg et al., J. Virol. 64:1556-1665, 1990). However, no IE86 binding sequences have been identified in the promoter. In addition, while HCMV-infected human foreskin fibroblasts showed a DNA binding activity specific for a pol promoter element termed IR1 (see Kerry et al., J. Virol. 68:4167-76, 1994), it is unclear which IE protein plays the central role in IR1 DNA binding activity.
While these and other studies have provided basic information about IE protein function, a greater understanding of the temporal cascade of viral gene expression is required in order to identify suitable targets for drug development. In particular, the identification of cell permissivity factors that are required for productive infection of host cells would provide a basis for the development of new therapeutic drugs. Such drugs are urgently needed for treatment of HCMV strains that are resistant to current therapies, which employ viral polymerase nucleoside analog inhibitors.
Accordingly, there is a need in the art for new therapies for HCMV infection targeting viral molecules necessary for the progression of the viral life cycle. The present invention fulfills these needs and further provides other related advantages.